CN103599569B - Preparation method of titanium alloy surface growth factor composite coating - Google Patents
Preparation method of titanium alloy surface growth factor composite coating Download PDFInfo
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- CN103599569B CN103599569B CN201310618763.8A CN201310618763A CN103599569B CN 103599569 B CN103599569 B CN 103599569B CN 201310618763 A CN201310618763 A CN 201310618763A CN 103599569 B CN103599569 B CN 103599569B
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Abstract
The invention relates to a preparation method of a titanium alloy surface growth factor composite coating. The preparation method comprises the steps of connecting growth factor nanogel and a target factor Biotin-PEG-NHS; then, treating the surface of a titanium alloy by using dopamine, and coupling Avidin onto the surface of the titanium alloy through the auto-agglutination action of the dopamine; then, fixing the growth factor nanogel on the surface of the titanium alloy through the special biological affinities of Biotin and Avidin, wherein growth factors include a bone morphogenetic protein (BMP), a transforming growth factor, a vascular endothelial growth factor, a platelet derived growth factor or fibroblast growth factor. The preparation method has the advantages of simplicity in reaction operation because of coupling the growth factor nanogel on the surface of the titanium alloy through simple one-pot reaction, mild in reaction condition and favorable in activity retention for a BMP-2.
Description
Technical field
The field of the present invention relates to is biological medical metal composite, growth factor nano gel is connected with targeting factor Biotin-PEG-NHS, then titanium alloy surface is used dopamine process, by the autohemagglutination effect of the free yl induction of dopamine, Avidin Avidin is coupled to titanium alloy surface, by the specific biocompatible effect of biotin Biotin and Avidin, growth factor nano gel is fixed to titanium alloy surface again, thus realizes a kind of preparation of new bio-medical bone alternate material
Background technology
Along with the trend of aged tendency of population and neurodegenerative disease rejuvenation, the sickness rate of various spinal column and joint diseases increases year by year, needs the quantity of carrying out spinal fusion surgery and joint replacement surgery also to increase progressively thereupon.Titanium alloy has excellent mechanical property and good biocompatibility, and it has been widely used in this type of operation as material of main part, and the market demand increases day by day.But because treatment target is the old people suffering from osteoporosis, the bone metabolism in their body is unbalance, and its bone resorption surpasses osteogenesis.And titanium alloy is biologically inert, itself and implant site osseointegration property are poor, are easy to surrounding host tissue and are separated and cause loosening.Therefore, improve the biological activity of titanium alloy, promote the repair ability of implant site osseous tissue, shorten the research focus that healing time becomes current medical titanium alloy, and the integration speed of titanium alloy and bone and the stability after implanting depend primarily on biological activity and the osteoinductive of face coat.
Based on the understanding to the Integrated implant process Middle molecule time, the somatomedin played a crucial role in the different phase of bone remoulding or the effect of gene clear and definite gradually.Increase implant site somatomedin concentration titanium alloy surface is growth factor-loaded, improve the formation speed of osseous tissue.As far back as 1989, insulin-like growth factor and titanium are just combined the reparation being used for bone by Aspenberg, and hereafter, some seminars have also carried out similar research successively both at home and abroad, at present for the application of orthopaedics, the bone morphogenetic protein 2 (BMP-2) of most study.The Li Zhongli of PLA General Hospital etc. adopt three-dimensional connected porous titanium structure composite BMP-2, and muscular grafting experiment finds that BMP-2 can impel fibrous tissue to grow into pore structure inside, stimulates angiogenesis, and finds that there is calcified bone organization formation after 30 days.
But for somatomedin, its practical application is all faced with release manner and active maintenance two subject matters.The titanium alloy surface growth factor delivery technology reported comprises: surface directly covalent coupling, by natural macromolecular covalent bond, the coating of biodegradable synthetic polymer physically trapping, hydroxyapatite and the carrying of titania nanotube reservoir type etc.Promote Integrated implant although somatomedin is applied to titanium alloy surface and repairs existing large quantifier elimination, being also difficult to meet clinical demand in activity maintenance and sustained-release etc.Recently, Lu Yun peak seminar is with Nanogel technology successful delivery somatomedin, and this technology, by radical reaction, with the coated albumen of macromolecule monomolecular film, forms the microgranule of particle diameter 40-80nm, effectively can improve the stability of albumen.Thus solve the problem of somatomedin stability.But when to titanium alloy material modification, due to the space steric effect that the inertia of titanium alloy itself and the large scale of somatomedin Nanogel cause, be difficult to realize the direct immobilization of somatomedin Nanogel at titanium alloy surface.Therefore suitable medium must be selected to realize this immobilization.
The present invention is based on and the process of growth factor nano gel surface is gone out targeting factor Biotin-PEG-NHS, then Avidin Avidin is fixed on titanium alloy surface by the mode of dopamine auto polymerization, realize the immobilization of growth factor nano gel at titanium alloy surface by the biocompatible effect of Biotin and Avidin, realize the function of bio-medical composition
Summary of the invention
The object of the invention is the growth factor nano gel with high stability to be coupled to titanium alloy surface by the dopamine auto polymerization reaction of free yl induction and the bio-identification effect of Biotin and Avidin, realize the quick reparation of implanting titanium alloy position osseous tissue.
Principle of the present invention is that the process of growth factor nano gel surface goes out targeting factor Biotin-PEG-NHS, then Avidin Avidin is fixed on titanium alloy surface by the mode of dopamine auto polymerization, realize the immobilization of growth factor nano gel at titanium alloy surface by the biocompatible effect of Biotin and Avidin, realize the function of bio-medical composition
Preparation method of the present invention is as follows:
The preparation method of titanium alloy surface growth factor composite coating, growth factor nano gel is connected with targeting factor Biotin-PEG-NHS, then titanium alloy surface is used dopamine process, by the autohemagglutination effect of dopamine, Avidin Avidin is coupled to titanium alloy surface, then by the specific biocompatible effect of biotin Biotin and Avidin, growth factor nano gel is fixed to titanium alloy surface
Described somatomedin comprises: bone morphogenetic protein (BMP), transforming growth factor (TGF), VEGF (VEGF), platelet derived growth factor (PDGF) or fibroblast growth factor (FGF).
Described targeting factor Biotin-PEG-NHS is Biotin-PEG
4-NHS and Biotin-PEG
20-NHS.
Described dopamine act as at the autohemagglutination of titanium alloy surface the radical polymerization that Ammonium persulfate. (APS) induces.
Build the preparation method of titanium alloy surface growth factor composite coating, step is as follows:
A. dopamine is dissolved in the PBS solution of 20mM pH=7.4 and is made into the dopamine solution that concentration is 1-4mg/ml, nitrogen current process 1-4h will be poured in above-mentioned solution; Polishing is carried out on Ti sheet surface, then respectively at normal hexane and 1-6mol/LN
asoak 30-60min in OH, then with milli-Q water to eluent in neutral; By the Ti sheet vacuum drying of gained; The titanium sheet handled well is joined in above-mentioned dopamine solution, then add 1-4mg Ammonium persulfate. (APS), under 30-60 DEG C of condition, react 2-6h;
B. the titanium sheet milli-Q water will obtained in a, is then soaked in the Avidin Avidin aqueous solution of 1-4mg/ml, reacts 1-24h under 20-40 DEG C of condition;
C. by growth factor nano gel dissolves in the borate buffer of 50mM pH=8.0, be prepared into the solution of 0.1-1mg/ml, get the Biotin-PEG-NHS solution that above-mentioned solution 1ml adds 1-10ul10mg/ml, magnetic agitation reaction 2-6h;
D. the titanium sheet milli-Q water of gained, natural drying under nitrogen flowing will be processed in b; Get gained solution 10-100ul in c and drip to titanium alloy surface, at 20-40 DEG C, react 6-12h.
Advantage of the present invention utilizes simple one pot reaction that growth factor nano gel is coupled to titanium alloy surface, and operation is simple, and reaction condition is gentle, has good activity keep for BMP-2.
Figure of description
The preparation method schematic diagram of titanium alloy surface growth factor composite coating;
Detailed description of the invention
Below by example, the present invention will be further elaborated.
Embodiment 1:
A. dopamine is dissolved in the PBS solution of 20mM pH=7.4 and is made into the dopamine solution that concentration is 1mg/ml, nitrogen current process 1h will be poured in above-mentioned solution; Polishing is carried out on Ti sheet surface, then respectively at normal hexane and 1-6mol/LN
asoak 30min in OH, then with milli-Q water to eluent in neutral; By the Ti sheet vacuum drying of gained; The titanium sheet handled well is joined in above-mentioned dopamine solution, then add 1mg Ammonium persulfate. (APS), under 30 DEG C of conditions, react 2h;
B. the titanium sheet milli-Q water will obtained in a, is then soaked in the Avidin Avidin aqueous solution of 1mg/ml, reacts 1h under 20 DEG C of conditions;
C. by growth factor nano gel dissolves in the borate buffer of 50mM pH=8.0, be prepared into the solution of 0.1mg/ml, get the Biotin-PEG-NHS solution that above-mentioned solution 1ml adds 1ul10mg/ml, magnetic agitation reaction 2h;
D. the titanium sheet milli-Q water of gained, natural drying under nitrogen flowing will be processed in b.Get gained solution 10ul in c and drip to titanium alloy surface, at 20 DEG C, react 6h;
Use ELISA kit to measure and be fixed on the amount of the somatomedin of titanium plate surface for 1.4ug.Titanium sheet is after gamma sterilization, and somatomedin still keeps the relative activity of 52%.
Embodiment 2:
A. dopamine is dissolved in the PBS solution of 20mM pH=7.4 and is made into the dopamine solution that concentration is 2mg/ml, nitrogen current process 2h will be poured in above-mentioned solution; Polishing is carried out on Ti sheet surface, then respectively at normal hexane and 3mol/L N
asoak 40min in OH, then with milli-Q water to eluent in neutral; By the Ti sheet vacuum drying of gained; The titanium sheet handled well is joined in above-mentioned dopamine solution, then add 2mg Ammonium persulfate. (APS), under 40 DEG C of conditions, react 4h;
B. the titanium sheet milli-Q water will obtained in a, is then soaked in the Avidin Avidin aqueous solution of 2mg/ml, reacts 12h under 30 DEG C of conditions;
C. by growth factor nano gel dissolves in the borate buffer of 50mM pH=8.0, be prepared into the solution of 0.5mg/ml, get the Biotin-PEG-NHS solution that above-mentioned solution 1ml adds 5ul10mg/ml, magnetic agitation reaction 4h;
D. the titanium sheet milli-Q water of gained, natural drying under nitrogen flowing will be processed in b.Get gained solution 50ul in c and drip to titanium alloy surface, at 30 DEG C, react 9h;
Use ELISA kit to measure and be fixed on the amount of the somatomedin of titanium plate surface for 3.2ug.Titanium sheet is after gamma sterilization, and somatomedin still keeps the relative activity of 36%.
Embodiment 3:
A. dopamine is dissolved in the PBS solution of 20mM pH=7.4 and is made into the dopamine solution that concentration is 4mg/ml, nitrogen current process 4h will be poured in above-mentioned solution; Polishing is carried out on Ti sheet surface, then respectively at normal hexane and 6mol/L N
asoak 60min in OH, then with milli-Q water to eluent in neutral; By the Ti sheet vacuum drying of gained; The titanium sheet handled well is joined in above-mentioned dopamine solution, then add 4mg Ammonium persulfate. (APS), under 30-60 DEG C of condition, react 6h;
B. the titanium sheet milli-Q water will obtained in a, is then soaked in the Avidin Avidin aqueous solution of 4mg/ml, reacts 24h under 40 DEG C of conditions;
C. by growth factor nano gel dissolves in the borate buffer of 50mM pH=8.0, be prepared into the solution of 1mg/ml, get the Biotin-PEG-NHS solution that above-mentioned solution 1ml adds 10ul10mg/ml, magnetic agitation reaction 6h;
D. the titanium sheet milli-Q water of gained, natural drying under nitrogen flowing will be processed in b.Get gained solution 100ul in c and drip to titanium alloy surface, at 40 DEG C, react 12h;
Use ELISA kit to measure and be fixed on the amount of the somatomedin of titanium plate surface for 2.3ug.Titanium sheet is after gamma sterilization, and somatomedin still keeps the relative activity of 73%.
Embodiment 4:
A. dopamine is dissolved in the PBS solution of 20mM pH=7.4 and is made into the dopamine solution that concentration is 2mg/ml, nitrogen current process 4h will be poured in above-mentioned solution; Polishing is carried out on Ti sheet surface, then respectively at normal hexane and 6mol/L N
asoak 60min in OH, then with milli-Q water to eluent in neutral; By the Ti sheet vacuum drying of gained; The titanium sheet handled well is joined in above-mentioned dopamine solution, then add 4mg Ammonium persulfate. (APS), under 60 DEG C of conditions, react 6h;
B. the titanium sheet milli-Q water will obtained in a, is then soaked in the Avidin Avidin aqueous solution of 4mg/ml, reacts 24h under 37 DEG C of conditions;
C. by growth factor nano gel dissolves in the borate buffer of 50mM pH=8.0, be prepared into the solution of 0.1mg/ml, get the Biotin-PEG-NHS solution that above-mentioned solution 1ml adds 10ul10mg/ml, magnetic agitation reaction 2h;
D. the titanium sheet milli-Q water of gained, natural drying under nitrogen flowing will be processed in b.Get gained solution 100ul in c and drip to titanium alloy surface, at 37 DEG C, react 6h;
Use ELISA kit to measure and be fixed on the amount of the somatomedin of titanium plate surface for 12.1ug.Titanium sheet is after gamma sterilization, and somatomedin still keeps the relative activity of 46%.
Embodiment 5:
A. dopamine is dissolved in the PBS solution of 20mM pH=7.4 and is made into the dopamine solution that concentration is 4mg/ml, nitrogen current process 1h will be poured in above-mentioned solution; Polishing is carried out on Ti sheet surface, then respectively at normal hexane and 2mol/L N
asoak 30min in OH, then milli-Q water to eluent is neutral; By the Ti sheet vacuum drying of gained; The titanium sheet handled well is joined in above-mentioned dopamine solution, then add 1mg Ammonium persulfate. (APS), under 30 DEG C of conditions, react 2h;
B. the titanium sheet milli-Q water will obtained in a, is then soaked in the Avidin Avidin aqueous solution of 1mg/ml, reacts 24h under 40 DEG C of conditions;
C. by growth factor nano gel dissolves in the borate buffer of 50mM pH=8.0, be prepared into the solution of 0.8mg/ml, get the Biotin-PEG-NHS solution that above-mentioned solution 1ml adds 8ul10mg/ml, magnetic agitation reaction 6h;
D. the titanium sheet milli-Q water of gained, natural drying under nitrogen flowing will be processed in b.Get gained solution 100ul in c and drip to titanium alloy surface, at 40 DEG C, react 12h;
Use ELISA kit to measure and be fixed on the amount of the somatomedin of titanium plate surface for 1.7ug.Titanium sheet is after gamma sterilization, and somatomedin still keeps the relative activity of 64%.
Claims (1)
1. a preparation method for titanium alloy surface growth factor composite coating, is characterized in that step is as follows:
A. dopamine is dissolved in the PBS solution of 20mM pH=7.4 and is made into the dopamine solution that concentration is 1-4mg/ml, nitrogen current process 1-4h will be poured in above-mentioned solution; Polishing is carried out on Ti sheet surface, then in normal hexane and 1-6mol/L NaOH, soaks 30-60min respectively, then with milli-Q water to eluent in neutral; By the Ti sheet vacuum drying of gained; The titanium sheet handled well is joined in above-mentioned dopamine solution, then add 1-4mg Ammonium persulfate. (APS), under 30-60 DEG C of condition, react 2-6h;
B. the titanium sheet milli-Q water will obtained in a, is then soaked in the Avidin Avidin aqueous solution of 1-4mg/ml, reacts 1-24h under 20-40 DEG C of condition;
C. by growth factor nano gel dissolves in the borate buffer of 50mM pH=8.0, be prepared into the solution of 0.1-1mg/ml, get the Biotin-PEG-NHS solution that above-mentioned solution 1ml adds 1-10ul 10mg/ml, magnetic agitation reaction 2-6h;
D. the titanium sheet milli-Q water of gained, natural drying under nitrogen flowing will be processed in b; Get gained solution 10-100ul in c and drip to titanium alloy surface, at 20-40 DEG C, react 6-12h;
Described somatomedin comprises: bone morphogenetic protein (BMP), transforming growth factor (TGF), VEGF (VEGF), platelet derived growth factor (PDGF) or fibroblast growth factor (FGF); Described targeting factor Biotin-PEG-NHS is Biotin-PEG
4-NHS and Biotin-PEG
20-NHS; Described dopamine act as at the autohemagglutination of titanium alloy surface the radical polymerization that Ammonium persulfate. (APS) induces.
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| CN107376036B (en) * | 2017-06-23 | 2020-01-07 | 淮阴工学院 | Construction method of enzyme response type multifunctional nano coating |
| CN107837418B (en) * | 2017-11-03 | 2020-06-02 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of biotinylation titanium plate loaded with adipose-derived stem cell microvesicles |
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